RESUMO
Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct in vivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.
Assuntos
Bactérias , Proteínas de Bactérias , Histidina Quinase , Viabilidade Microbiana , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Elétrons , Histidina Quinase/genética , Histidina Quinase/metabolismo , Microscopia de Polarização , Nitratos , Óperon/genética , Fosforilação , Regiões Promotoras Genéticas , Multimerização Proteica/efeitos dos fármacos , Análise de Célula Única , Fatores de TempoRESUMO
Traumatic injury resulting in hemorrhage is a prevalent cause of death worldwide. The current standard of care for trauma patients is to restore hemostasis by controlling bleeding and administering intravenous volume resuscitation. Adequate resuscitation to restore tissue blood flow and oxygenation is critical within the first hours following admission to assess severity and avoid complications. However, current clinical methods for guiding resuscitation are not sensitive or specific enough to adequately understand the patient condition. To better address the shortcomings of the current methods, an approach to monitor intestinal perfusion and oxygenation using a multiwavelength (470, 560, and 630 nm) optical sensor has been developed based on photoplethysmography and reflectance spectroscopy. Specifically, two sensors were developed using three wavelengths to measure relative changes in the small intestine. Using vessel occlusion, systemic changes in oxygenation input, and induction of hemorrhagic shock, the capabilities and sensitivity of the sensor were explored in vivo. Pulsatile and nonpulsatile components of the red, blue, and green wavelength signals were analyzed for all three protocols (occlusion, systemic oxygenation changes, and shock) and were shown to differentiate perfusion and oxygenation changes in the jejunum. The blue and green signals produced better correlation to perfusion changes during occlusion and shock, while the red and blue signals, using a new correlation algorithm, produced better data for assessing changes in oxygenation induced both systemically and locally during shock. The conventional modulation ratio method was found to be an ineffective measure of oxygenation in the intestine due to noise and an algorithm was developed based on the Pearson correlation coefficient. The method utilized the difference in phase between two different wavelength signals to assess oxygen content. A combination of measures from the three wavelengths provided verification of oxygenation and perfusion states, and showed promise for the development of a clinical monitor.
Assuntos
Jejuno , Monitorização Fisiológica/instrumentação , Oximetria/instrumentação , Oxigênio/sangue , Processamento de Sinais Assistido por Computador , Algoritmos , Animais , Pressão Sanguínea/fisiologia , Desenho de Equipamento , Jejuno/irrigação sanguínea , Jejuno/fisiologia , Jejuno/cirurgia , Oximetria/métodos , Fotopletismografia/instrumentação , Coelhos , Fluxo Sanguíneo Regional/fisiologia , Choque Hemorrágico/sangue , Choque Hemorrágico/diagnósticoRESUMO
The quantification of visceral organ oxygenation after trauma-related systemic hypovolemia and shock is critical to enable effective resuscitation. In this work, a photoplethysmography-based (PPG) sensor was specifically designed for probing the perfusion and oxygenation condition of intestinal tissue with the ultimate goal to monitor patients post trauma to guide resuscitation. Through Monte Carlo modeling, suitable optofluidic phantoms were determined, the wavelength and separation distance for the sensor was optimized, and sensor performance for the quantification of tissue perfusion and oxygenation was tested on the in-vitro phantom. In particular, the Monte Carlo simulated both a standard block three-layer model and a more realistic model including villi. Measurements were collected on the designed three layer optofluidic phantom and the results taken with the small form factor PPG device showed a marked improvement when using shorter visible wavelengths over the more conventional longer visible wavelengths. Overall, in this work a Monte Carlo model was developed, an optofluidic phantom was built, and a small form factor PPG sensor was developed and characterized using the phantom for perfusion and oxygenation over the visible wavelength range. The results show promise that this small form factor PPG sensor could be used as a future guide to shock-related resuscitation.